Two novel neuropeptides, orexin-A and orexin-B, are identified as endogenous ligands of two orphan G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). Orexins are implicated in regulating a variety of physiological processes, such as wake/sleep states, feeding behaviors etc. A previous work (Liu et al, 2011) in this laboratory revealed the expression of orexin-A and orexin-B in rat retinal neurons. In the present work we investigated how orexin-A modulated current responses mediated by AMPA receptor (AMPAR) of ganglion cells (GCs) and amacrine cells (ACs) in rat retina. Animal treatments in this work coincide with the NIH guidelines for animal experimentation and the guidelines of Fudan University on the ethical use of animals. Male Sprague-Dawley rats (P15-20 d) were anaesthetized by intraperitoneal injection with 25% urethane (0.4 ml/100 g) and killed by decollation. By immunofluorescence double labeling, we first showed that OX1R was mainly expressed in GCs, whereas OX2R was present in both ACs and GCs. Using whole-cell patch clamp techniques, we further examined effects of orexin-A on AMPAR-mediated currents recorded from isolated GCs and ACs and explored mechanisms underlying these effects. Administration of orexin-A suppressed AMPAR-mediated currents in GCs, and the effect was abolished by co-application of SB334867, a selective OX1R antagonist, but not by TCSOX229, a selective OX2R blocker. In contrast, orexin-A increased AMPA responses of ACs, which could be reversed by TCSOX229, but not by SB334867. As expected, either preincubation with the Gi/o inhibitor pertussis toxin or intracellular dialysis of the Gi/o activator mastoparan eliminated both the orexin-A effects on GCs and ACs. Signaling pathways mediating the two effects were further explored. Firstly, the two effects involved activation of PKC, as evidenced by the elimination of those by PKC antagonists. Secondly, U73122, a phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, and D609, a phosphatidylcholine (PC)-PLC inhibitor, abolished the effects on AMPA currents of GCs and ACs respectively. Thirdly, the two effects were different in utilizing intracellular Ca2+. Whilst the effect on GCs may be mediated by the release of intracellular Ca2+ through the IP3-sensitive pathway, that on ACs was independent of intracellular Ca2+. Furthermore, neither cAMP-PKA nor cGMP-PKG pathway was involved in both the effects. In consequence, a distinct PI-PLC/IP3/Ca2+/PKC signaling pathway, following the activation of OX1R, is most likely responsible for the orexin-A-induced suppression of AMPA currents of rat GCs, whereas a PC-PLC/PKC signaling pathway, following the activation of OX2R, mediates the orexin-A-induced potentiation of AMPA currents of ACs.